Not Applicable
The present invention relates to methods for increasing the dynamic range of mass spectrometers. More specifically, the present invention is a method of improving the performance of mass spectrometers by first generating a mass spectrum, and then trapping and selectively ejecting ions using resonant rf excitation in a quadrupole ion filter based upon information from the prior spectrum.
Progress in a wide range of scientific inquiry requires the qualitative and quantitative analysis of molecules, and important classes of problems involve the analysis of complex mixtures where the relative abundances of mixture components vary over many orders of magnitude. For example, a major goal of biological research in the field of proteomics is the understanding of protein functions in a cellular context. Unfortunately, many important protein classes necessary for this understanding are present only at low concentrations. As noted in Godovac-Zimmerman, J.; Brown, L. Mass Spectrom. Rev., 2000, 20, 1-57, the range of peptide (or protein) concentrations of interest in proteomic measurements can vary more than six orders of magnitude and can include  greater than 105 components. When analyzed in conjunction with capillary LC separations, both the total ion production rate from ESI and the complexity of the mixture at any point can vary by more than two orders of magnitude, and the relative abundances of specific components of interest can vary by  greater than 106. This variation in ion production rate and spectral complexity constitutes a major challenge for proteome analyses. For example, the elution of highly abundant peptides can restrict the detection of lower-level co-eluting peptides since the dynamic range presently achieved in a single spectrum is xcx9c103 for a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer and  less than  or xcx9c102 for an ion trap mass spectrometer. If the ion accumulation process (i.e., ion accumulation time) is optimized for the most abundant peaks, the accumulation trap will not be filled to capacity during the elution of lower abundance components from a chromatographic or electrophoretic separation, and the overall experimental dynamic range will be significantly constrained. If, however, longer accumulation times are used, the conditions conventionally used result in an xe2x80x9coverfillingxe2x80x9d of the analyzer trap in many cases, which will be manifested by biased accumulation, loss of measurement accuracy, or extensive activation and dissociation of the analytes. Thus, there is a need for methods aimed at avoiding the undesired artifacts associated with overfilling the mass analyzer trap. There is a further need for approaches which will also simultaneously expand the dynamic range of measurements.
Those having skill in the art have proposed a variety of methods and techniques to expand this dynamic range. In one such approach, a quadrupole ion filter is used as some combination of high and low bandpass filters, i.e. a mass filter to select a specific species or mass range for detailed analysis. However, this approach is targeted at a specific m/z peak or limited mass range and results in the loss of possible information on other low abundance species, and this is not generally useful in the characterization of complex mixtures. Therefore, there exists a need for methods for enhancing the dynamic range of mass spectrometers that can address complex mixtures with components having abundances spanning many orders of magnitude.
Accordingly, the present invention is a method for increasing the dynamic range of mass spectrometers. More particularly, the present invention finds particular utility in increasing the dynamic range of mass spectrometers which utilize ion trap type mass analyzers, such as quadrupole ion trap mass spectrometers (ITMS) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers. By way of example, and not meant to be limiting, when analyzing complex protein digests, the present invention can increase the dynamic range of a mass spectrometer through the simultaneous and selective suppression of higher abundance peaks dispersed cross the mass spectrum. By eliminating these ions, lower abundant species can be analyzed since they can be accumulated to detectable levels, resulting in an increase in the dynamic range of the instrument.
As practiced by the present invention, selective ejection of the most abundant ion species from a quadrupole filter, is performed with rf excitation. Such excitation can be dipolar, quadrupolar, or parametric. If the frequency of the auxiliary rf-field is equal to the secular frequency (i.e., resonant excitation) or to the doubled secular frequency (i.e., parametric excitation) of a particular m/z ion species, the auxiliary rf-field causes these ions to oscillate with increased amplitudes. By introducing a supplemental rf-field, ions stored in a quadrupole ion filter can thus be efficiently ejected using either parametric excitation or resonant excitation.
The present invention thus increases the dynamic range of a mass spectrometer by utilizing a quadrupole ion filter as a device to selectively remove one or more undesired ions (peaks), thereby allowing the accumulation and subsequent detection of desired ions in a mass analyzer, such as an ion trap operated as a mass analyzer, adjunct to the ion filter. Typically, but not meant to be limiting, the desired ions are those that are present at relatively low concentrations, while the undesired ions are those that are present at relatively high concentrations. Accordingly, the present invention finds particular utility in instruments where ion capacity is constrained, such as mass spectrometers which utilize ion trapping in their analysis and detection schemes.
The method of the present invention first passes a sample of ions through the mass spectrometer having a quadrupole ion filter, whereupon the intensities of the mass spectrum of the sample are measured. From the mass spectrum, ions within this sample are then identified for subsequent ejection. Typically, the ions identified for subsequent ejection will be the most highly abundant species, as the ejection of these species produces the most additional xe2x80x9croomxe2x80x9d for further accumulation in the ion trap. However, it may not always be the case that ions are selected for ejection based purely on their abundance. In certain applications, ions are selected simply because they are not of interest to the desired analysis, even though they are not the most abundant. The present invention should thus be broadly construed to include any application where ions are selectively ejected using rf excitation to make room for further accumulation.
As further sampling introduces ions into the mass spectrometer, the appropriate rf voltages are applied to a quadrupole ion filter, thereby selectively ejecting the undesired ions previously identified. In this manner, the desired ions may be collected for longer periods of time in the mass analyzer, thus allowing better collection and subsequent analysis of the desired ions.
The mass analyzer used for accumulation may be the same ion trap used for mass analysis in a FTICR or ITMS, in which case the mass analysis is performed directly, or it may be an intermediate trap. In the case where collection is an intermediate trap, the desired ions are accumulated in the intermediate trap, and then transferred to a separate ion trap in a FTICR or ITMS, where the mass analysis is performed.
The method of the present invention may be further enhanced as follows. Those skilled in the operation of ion trapping mass spectrometers generally have an understanding of the optimal level of charge, or ions, that can be introduced into a given trap, without causing undesirable effects on ion identification. Accordingly, when practicing the method of the present invention with a given sample of some unknown, a skilled artisan, utilizing a computer controlled series of steps, would first determine the amount of time necessary to fill the ion trap within the instrument to some optimal level of ions. The proportion of the ions that were then identified for selective ejection (the undesired ions) would then be compared to the total mass spectrum. In that manner, the skilled artisan could accurately gauge the length of time necessary to fill the ion trap to its"" optimal level with the desired ions, while ejecting undesired ions in the ion filter in the manner described above. As further introduction of ions proceeded, with the ejection of those undesired ions identified in the initial evaluation, the ion trap can be easily filled to the optimal level with only the desired ions, including many that likely were not detectable before this step.
If, by way of example, it were determined that 90% of the ions in a given sample were undesirable ions to be ejected, then ten times the initial amount of time needed to fill the ion trap would be allowed to pass while ejecting those undesirable ions. In this manner, the ion trap is filled to the optimal level with only desirable ions. Those having skill in the art will recognize that the precise amount of time necessary to fill the trap becomes a function of the optimal level to which the trap is filled, and the proportion of a given sample that is to be ejected according to the method of the present invention. Suitable adjustments for any particular circumstance can thus be made to optimize the instruments performance, and this type of control can readily be accomplished by the computer that acquires data during mass spectrum analysis.
As will be apparent to those having skill in the art, the method of the present invention can further be repeated as many times as desired to achieve ever greater dynamic range for the instrument. For example, several undesirable species may be identified and eliminated as described above. As noted above, those will typically be species that are highly abundant. However, xe2x80x9cabundancexe2x80x9d is a relative term. Once those ions that were highly abundant in the initial sampling are removed, a different set of ions will predominate, and new ions that were previously undetectable will appear. A portion of these ions may then further be identified as undesirable. In addition to the highly abundant species previously identified, a portion of these ions may also be eliminated in subsequent trapping and analysis, using the same technique of rf excitation at the appropriate level for each identified ion. In this manner, the dynamic range of the instrument can be expanded in a step-wise fashion to theoretically infinite levels.
The operation and use of the present invention is more fully illustrated in the description of the preferred embodiments and the experiments conducted to demonstrate the efficacy of the present invention that follow. However, the specific examples set forth in the description of the preferred embodiments and the experiments should in no way be construed as limiting the scope of the present invention in its broader aspects, and the present invention should be understood to encompass and include any and all variations and combinations of any specific equipment that might be utilized to accomplish the basic steps set forth in the summary of the invention provided herein.